Biology subjects

Hernandez, C. J.

Publications and source records attributed to Hernandez, C. J..

15 recordsLinked to original sources

bioRxiv2026

Mechanofiltration Enables High-Throughput Measurements of Bacterial Cell Mechanics

Bacteria experience diverse mechanical forces throughout their natural environments, yet quantitative measurements of bacterial biomechanics remain challenging because most existing techniques require specialized instrumentation, such as atomic force microscopy (AFM) or microfluidic devices. Here, we introduce mechanofiltration, a simple, high-throughput assay that estimates whole-cell mechanics using standard laboratory equipment. In mechanofiltration, bacterial suspensions are centrifuged through porous membranes in multiwell plates. Pressure generated during centrifugation drives cells toward pores that are smaller than cell width, requiring cells to deform to transit through the membrane. By combining recovery on the opposite side of the filter with measurements of cell size, the assay estimates maximum cell deformation, a size-corrected proxy for whole-cell stiffness. We show that mechanofiltration detects established reductions in Escherichia coli cell stiffness caused by genetic disruption of load-bearing cell envelope components, treatment with outer membrane-destabilizing agents, and sublethal exposure to antibiotics. Fold changes in maximum deformation closely agree with published measurements obtained using AFM, cell-bending assays, and osmotic shock experiments. Mechanofiltration also reproduces previously reported mechanical differences among bacterial species with distinct morphologies and envelope architectures. Together, these results establish mechanofiltration as an accessible, inexpensive, and scalable approach for identifying genetic and environmental determinants of bacterial mechanics and for high-throughput screening of bacterial biomechanical phenotypes.

microbiology↗

bioRxiv2026

Melanopsin regulates axonal translation underlying retinohypothalamic circuit assembly

Intrinsically photosensitive retinal ganglion cells (ipRGCs) influence visual system development via melanopsin before photoreceptor-mediated vision, but how melanopsin signaling contributes to ipRGC circuit assembly remains unknown. Here we show that melanopsin coordinates retinohypothalamic tract development by regulating local translation in developing ipRGC axons. Loss of melanopsin selectively disrupted local translation in axons without affecting somatic translation. The affected transcripts encoded cytoskeletal regulators, adhesion molecules, and trafficking proteins, and activity-dependent changes in translation were restricted to the period before eye-opening. Consistent with impaired axonal growth and synaptogenesis, Opn4 knockout mice showed reduced ipsilateral suprachiasmatic nucleus innervation and fewer retinohypothalamic synapses, while nanoscale synaptic molecular organization and microglial engulfment were unaffected. Reduced visual drive in Opn4 knockouts further altered developmental gene expression programs across the retina, suprachiasmatic nucleus, and lateral geniculate nucleus, with region-specific differences in expression timing. These findings identify melanopsin as a regulator of local axonal translation during early circuit development, linking sensory phototransduction to translational control mechanisms that guide retinohypothalamic tract assembly and postsynaptic target maturation.

neuroscience↗

bioRxiv2026

Gut microbiome-dependent IL-1 signaling is a mediator of ACVR1R206H-driven heterotopic ossification

Inflammatory diseases cause significant morbidity and mortality, but their pathobiology is often difficult to dissect due to complex genetic-environmental interactions. Genetic forms of heterotopic ossification, such as fibrodysplasia ossificans progressiva (FOP), reduce genetic variability, allowing careful dissection of non-genetic drivers of inflammation. While >95% of FOP patients harbor the ACVR1R206H mutation, patients exhibit significant variability in disease progression, suggesting a role of environmental drivers. Here, we identify the gut microbiome as a regulator of inflammation-driven HO in FOP. Metagenomic profiling of cohabitating FOP/unaffected sibling pairs revealed a pathogenic gut microbiome profile in FOP patients (Bray-Curtis, p < 0.05). In Pdgfr-Cre/Acvr1R206H (FOP) mice, gut microbiome ablation by antibiotics reduced spontaneous HO formation (47.4% reduction, p < 0.05) and reduced plasma IL-1 pathway activity. IL-1{beta} blockade in FOP mice suppressed trauma-induced HO formation. These findings identify a gut microbiome-IL-1-HO axis with modifiable targets for developing treatments for HO and related inflammatory conditions. One Sentence SummaryAntibiotic disruption of the gut microbiome reduces HO in FOP mice via an IL-1 mediated pathway.

immunology↗

bioRxiv2025

Modifications to the gut microbiome alter bone matrix proteomics and fracture toughness at the cellular scale

The gut microbiome can regulate the strength of bone matrix but the specific changes in matrix function and composition are not yet understood. Here we introduce micropillar splitting to determine the fracture toughness of matrix at the cellular scale in concert with proteomic analysis of bone matrix when the gut microbiome was altered by oral antibiotics (ampicillin+neomycin). Male mice were divided into four groups (n = 3-4/group): (1) Unaltered (no alteration), (2) Continuous (alteration from 4-24 weeks), (3) Delayed (alteration from 16-24 weeks) and (4) Reconstituted (alteration from 1-16 weeks following by reconstitution). Micropillars (5 {micro}m diameter) were fabricated using focused ion beam milling on femur cross-sections in regions of matrix formed either before or after changes in the microbiome (16 weeks) (n = 4/group). Proteomics was used to identify differences in matrix protein composition. Micropillar fracture toughness differed by group (p < 0.001) and region (p < 0.001). Fracture toughness in the Unaltered group (1.34 {+/-} 0.32 MPa{surd}m, mean {+/-} SD) was substantially greater than the Continuous group (0.95 {+/-} 0.20) and the Delayed group (0.90 {+/-} 0.21) but not different from the Reconstituted group (1.22 {+/-} 0.25). Bone matrix formed from 16-24 weeks of age had lower fracture toughness than matrix formed before 16 weeks of age in all groups. Notably, micropillar splitting was substantially more precise than whole bone testing; whole bone notched 3-point bending tests did not detect differences in fracture toughness. Proteomics identified 46 extracellular matrix proteins that were differentially abundant between groups, including decreased abundance of Periostin (q < 0.001) and Emilin-1 (q < 0.001) in groups with impaired bone matrix. These findings demonstrate that modifications to the gut microbiome lead to changes in bone matrix throughout cortical bone volume and establish micropillar splitting as a high-precision approach for characterizing matrix material properties. Plain Language SummaryThis study investigated the effect of the gut microbiome on the brittleness of bone as a material. We used a new technique for measuring the brittleness of bone that involves making microscopic pillars on the bone surface and splitting them down the middle to measure a material property called fracture toughness. Mice with altered gut microbiomes had bone with reduced fracture toughness (by 35-40%). Restoration of an altered gut microbiome for two months reversed the effects. The effect of the microbiome on bone occurred throughout the whole bone and was not limited to regions of the bone formed after a change in the gut microbiome.

bioengineering↗

bioRxiv2025

A load-bearing function for the cytoplasmic membrane of Escherichia coli

The structural integrity of bacterial cells is traditionally attributed to the peptidoglycan cell wall, and more recently to the outer membrane, with the cytoplasmic membrane assumed to be mechanically passive. Cells lacking filaments of the actin homolog MreB are more bendable, suggesting a role for the cytoskeleton in cell stiffness. Here, we show that MreB does not stiffen the envelope directly, but instead mechanically couples the cell wall to the cytoplasmic membrane through its role in peptidoglycan synthesis, increasing resistance to bending. Under hyperosmotic stress, MreB relocalized to the poles, forming linkages that prevent membrane detachment from the cell wall and attenuate cytoplasmic contraction. Disruption of MreB filament formation, nutrient starvation, or inactivation of glycan elongation factors abolished or reduced this coupling, revealing that peptidoglycan biosynthesis actively mediates stress distribution across surface layers. Our findings redefine the bacterial envelope as a mechanically integrated composite, with the cytoplasmic membrane having substantial load-bearing capacity.

microbiology↗

bioRxiv2025

Commensal taxa in gut microbiota limit antibiotic resistance during extended oral antibiotic use

Certain bacterial infections, such as those involving prosthetics, can require antimicrobial therapy over months to years, potentially increasing the burden of antimicrobial resistance. Here we longitudinally track the antimicrobial resistome in mice during continuous antibiotic dosing over 21 months. The burden of antibiotic resistance genes (ARGs) initially increases, but, surprisingly, declines in later months, approaching levels observed in untreated animals. ARG burden is regulated by taxonomy and declines as ARG-harboring taxa that initially bloom are replaced by commensals. Furthermore, we find that the dynamics of antibiotic-induced ARG burden are influenced by age-related differences in microbial taxonomy and can be removed by fecal microbiota transplantation. We show that commensals may regulate the resistome by limiting the growth of ARG-harboring taxa, thereby providing antimicrobial expansion resistance.

microbiology↗

bioRxiv2025

Physical mechanisms that enable bacteria to traverse channels half their width

Bacteria colonize surfaces in the environment and can also penetrate tissues and materials by entering micro- and nanoscale cracks and pores. Staphylococcus aureus has been observed within nanoscale channels in bone that are 2-3 times smaller than cell diameter. Once inside bone, bacteria are protected from host immunity and systemic antibiotics, potentially contributing to chronic and recurrent infections. The physical mechanisms that enable bacteria to enter channels smaller than cell width are unclear. It has been proposed that bacteria traverse narrow passages through division - such that daughter cells form within small channels and eventually create a chain of cells extending down the channel length. Here we use microfluidics to test the idea that S. aureus can traverse bone-like nanochannels through growth. We examined 322 individual cells trapped within tapered nanochannels ([~]1.5 to 0.4 mm in width). When cells were deformed below 606 nm (65% original width), growth and division were slowed or completely inhibited. When cell division did occur in nanochannels, daughter cells were more likely to travel towards the wider side of the channel. Hence, it is unlikely that cell division would preferentially enable transit of S. aureus into nanoscale channels. However, the magnitudes of fluid pressure needed to deform S. aureus to widths similar to that seen in bone (1 to 6.5 kPa), were small relative to fluid pressure in bone generated by physical forces in vivo (8-20 kPa). Thus, our findings suggest that colonization of nanoscale channels in bone or other tissues is more likely due to moderate fluid pressure rather than growth. ImportanceBacteria that colonize materials and tissues within the body can be difficult to remove, even with thorough cleaning and application of antibiotics. Recent studies show that bacteria not only colonize the surfaces of tissues in the body but can also squeeze into naturally occurring pores and channels and thereby gain protection from immune cells and antibiotics. Here we ask how physical forces and cell growth might enable bacteria to enter small pores within materials. We use microfluidic devices to study the growth and migration of the human pathogenic bacteria, S. aureus, which is the leading cause of chronic bone infections.

microbiology↗

bioRxiv2025

Dietary Menaquinone-9 Supplementation Does Not Influence Bone Tissue Quality or Bone Mineral Density in Mice

Vitamin K has been implicated in skeletal health because vitamin K-dependent proteins are present in bone. While there are multiple forms of vitamin K, most research has focused on phylloquinone, which is found mainly in plant-based foods, and its metabolite menaquinone-4 (MK4). However, there are additional forms of vitamin K that are bacterially produced that appear to influence bone health but have not yet been studied extensively. Herein, we evaluated the effects of menaquinone-9 (MK9), a bacterially produced form of vitamin K on bone tissue quality and density in young mice. Four-week-old male (n=32) and female (n=32) C57BL/6 mice were supplemented with 0.06 mg/kg diet or 2.1 mg/kg diet of MK9 for 12 weeks. During week 11, a sub-group of mice (n=7/sex/group) received daily deuterium-labeled MK9 to trace its metabolic fate in bone. Liver MK4 and MK9 were significantly higher in mice fed 2.1 mg MK9/kg compared to those receiving 0.06 mg MK9/kg, regardless of sex (all p [≤] 0.017). MK4 was the only vitamin K form detected in bone, with 63-67% of skeletal MK4 in mice fed 2.1 mg MK9/kg derived from deuterium-labeled MK9. Femoral tissue strength, maximum bending moment, section modulus, and bone mineral density did not differ significantly across diet groups in either sex (all p[≥]0.083). Cross-sectional area (p=0.003) and moment of inertia (p=0.001) were lower in female mice receiving 2.1 mg MK9/kg compared to those receiving 0.06 mg MK9/kg, but no differences were found in male mice. Higher bone MK4 concentrations did not correlate with higher bone tissue quality or density. Despite dietary MK9 being a dietary precursor to MK4 in bone, dietary MK9 supplementation did not affect bone tissue quality or bone mineral density. Lay summaryMost research about vitamin K and bone health has focused on phylloquinone, the plant-based vitamin K form, and its metabolite menaquinone-4. Because interest in bacterially produced forms of vitamin K, which are abundant in the intestinal microbiome, is growing, we evaluated the effect of menaquinone-9 (a bacterially-produced form of vitamin K) on skeletal health. We supplemented mice with low and high doses of menaquinone-9 and also used stable-isotope labeled menaquinone-9 to trace its conversion to menaquinone-4 in bone. We found menaquinone-9 served as a precursor to menaquinone-4 in bone, but menaquinone-9 supplementation did not improve bone health.

physiology↗

bioRxiv2024

Combinatorial characterization of bacterial taxa-driven differences in the microbiome of oyster reefs

Oyster reefs are invaluable ecosystems that provide a wide array of critical ecosystem services, including water filtration, coastal protection, and habitat provision for various marine species. However, these essential habitats face escalating threats from climate change and anthropogenic stressors. To combat these challenges, numerous oyster restoration initiatives have been undertaken, representing a global effort to preserve and restore these vital ecosystems. A significant, yet poorly understood, component of oyster reefs is the microbial communities. These communities account for a substantial proportion of marine reefs and are pivotal in driving key biogeochemical processes. Particularly, the environmental microbiome plays a crucial role in supporting the health and resilience of oyster populations. In our study, we sought to shed light on the microbiome within oyster reef ecosystems by characterizing the abundance, and diversity of microorganisms in the soil, biofilm, and oysters in 4 sites using a combinatorial approach to identify differentially abundant microbes by sample type and by sampling location. Our investigation revealed distinct microbial taxa in oysters, sediment and biofilm. The maximum Shannon Index indicated a slightly increased diversity in Herons Head (5.47), followed by Brickyard park (5.35), Dunphy Park (5.17) and Point Pinole (4.85). This is likely to be driven by significantly higher oyster mortality observed at Point Pinole during routine monitoring and restoration efforts. Interestingly Ruminococcus, Streptococcus, Staphylococcus, Prevotella, Porphyromonas, Parvimonas, Neisseria, Lactococcus, Haemophilus, Fusobacterium, Dorea, Clostridium, Campylobacter, Bacteroides, and Akkermansia were positively associated with the biofilm. Yet we have limited understanding of their beneficial and/or detrimental implications to oyster growth and survival. By unraveling the intricate relationships in microbial composition across an oyster reef, our study contributes to advancing the knowledge needed to support effective oyster reef conservation and restoration efforts.

microbiology↗

bioRxiv2024

Determining the Youngs Modulus of the Bacterial Cell Envelope

Bacteria experience substantial physical forces in their natural environment including forces caused by osmotic pressure, growth in constrained spaces, and fluid shear. The cell envelope is the primary load-carrying structure of bacteria, but the mechanical properties of the cell envelope are poorly understood; reports of Youngs modulus of the cell envelope of E. coli are widely range from 2 MPa to 18 MPa. We have developed a microfluidic system to apply mechanical loads to hundreds of bacteria at once and demonstrated the utility of the approach for evaluating whole-cell stiffness. Here we extend this technique to determine Youngs modulus of the cell envelope of E. coli and of the pathogens V. cholerae and S. aureus. An optimization-based inverse finite element analysis was used to determine the cell envelope Youngs modulus from observed deformations. The Youngs modulus of the cell envelope was 2.06 {+/-} 0.04 MPa for E. coli, 0.84 {+/-} 0.02 MPa for E. coli treated with a chemical known to reduce cell stiffness, 0.12 {+/-} 0.03 MPa for V. cholerae, and 1.52 {+/-} 0.06 MPa for S. aureus (mean {+/-} SD). The microfluidic approach allows examining hundreds of cells at once and is readily applied to Gram-negative and Gram-positive organisms as well as rod-shaped and cocci cells, allowing further examination of the structural causes of differences in cell envelope Youngs modulus among bacteria species and strains.

bioengineering↗

bioRxiv2024

Microbiome-induced Increases and Decreases in Bone Tissue Strength can be Initiated After Skeletal Maturity

Recent studies in mice have indicated that the gut microbiome can regulate bone tissue strength. However, prior work involved modifications to the gut microbiome in growing animals and it is unclear if the same changes in the microbiome, applied later in life, would change matrix strength. Here we changed the composition of the gut microbiome before and/or after skeletal maturity (16 weeks of age) using oral antibiotics (ampicillin + neomycin). Male and female mice (n=143 total, n=12-17/group/sex) were allocated into five study groups:1) Unaltered, 2) Continuous (dosing 4-24 weeks of age), 3) Delayed (dosing only 16-24 weeks of age), 4) Initial (dosing 4-16 weeks of age, suspended at 16 weeks), and 5) Reconstituted (dosing from 4-16 weeks following by fecal microbiota transplant from Unaltered donors). Animals were euthanized at 24 weeks of age. In males, bone matrix strength in the femur was 25-35% less than expected from geometry in mice from the Continuous (p= 0.001), Delayed (p= 0.005), and Initial (p=0.040) groups as compared to Unaltered. Reconstitution of the gut microbiota, however, led to a bone matrix strength similar to Unaltered animals (p=0.929). In females, microbiome-induced changes in bone matrix strength followed the same trend as males but were not significantly different, demonstrating sex-related differences in the response of bone matrix to the gut microbiota. Minor differences in chemical composition of bone matrix were observed (Raman spectroscopy). Our findings indicate that microbiome-induced impairment of bone matrix in males can be initiated and/or reversed after skeletal maturity. The portion of the femoral cortical bone formed after skeletal maturity (16 weeks) is small; however, this suggests that microbiome-induced changes in bone matrix occur without osteoblast/osteoclast turnover using an, as of yet unidentified mechanism. These findings add to evidence that the mechanical properties of bone matrix can be altered in the adult skeleton.

bioengineering↗

bioRxiv2024

Effects of long-term high dose aspartame on body mass, bone strength, femoral geometry, and microbiota composition in a young and aged cohort of male and female mice

BackgroundRecent reassessment of the safety of aspartame has prompted increased evaluation of its effect on the health of a range of tissues. The gut microbiome is altered by oral aspartame. One prior study suggested that changes in the microbiome caused by aspartame could influence the strength of bone in young skeletally developing mice. Here we ask how aspartame influences bone in mice of different age and sex. ObjectiveThe objective of this study was to determine the effect of aspartame on the bone strength and gut microbiota of young and aged mice. MethodsMale and female C57Bl/6J mice were untreated or treated with a high dose of aspartame in their drinking water from 1 month of age until 4 (young cohort; n = 80) or 22 months (aged cohort; n = 52). ResultsIn aged males, mice treated with aspartame had greater body mass, whole bone strength, and femoral geometry relative to untreated. Specifically, in aged males, aspartame led to 9% increase in body mass (p < 0.001), 22% increase in whole bone strength (p = 0.006), and 17% increase in section modulus (p < 0.001) relative to untreated mice. Aged males and females receiving aspartame had a different microbiota than untreated mice and a decreased abundance of Odoribacter. No differences in body mass, whole bone strength, or femoral geometry were associated with aspartame dosing in young males or young or aged females. ConclusionsAspartame treated aged males had greater whole bone strength and the effect appeared to be explained by greater body mass. Aspartame treatment did not alter whole bone strength in young males or young or aged females despite the aspartame having a similar effect on the microbiota of both aged males and females.

bioengineering↗

bioRxiv2023

Solute Transport in Engineered Living Materials using Bone Inspired Microscale Channel Networks

Engineered living materials (ELMs) are an emerging class of materials that is synthesized and/or populated by living cells. Maintaining living cells within an ELM over prolonged periods remains a major technical challenge that limits the service life of a material. Biological materials regularly maintain robust populations of living cells. Bone maintains living cells for decades by delivering nutrients through a network of nanoscale channels punctuated by microscale pores. Nutrient transfer in bone is enabled by mechanical loading experienced during regular use. Here we identify the characteristics of channel-pore network geometries and external mechanical loading that can be used in engineered living materials to deliver nutrients to resident cell populations. Transport occurs when deformation in the microscale pore network exceeds the volume of the connecting channels. Computational models show that transport is enhanced at greater load magnitudes and lower loading frequencies and are consistent with experimental validation using microfluidic systems. Our findings provide quantitative design principles for channel-pore networks capable of delivering nutrients to materials designed to house living cells.

bioengineering↗

bioRxiv2023

Deficiency of macrophage-derived Dnase1L3 causes lupus-like phenotypes in mice

Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease caused by environmental factors and loss of key proteins. One such protein is a serum endonuclease secreted by macrophages and dendritic cells, Dnase1L3. Loss of Dnase1L3 causes pediatric-onset lupus in humans is Dnase1L3. Reduction in Dnase1L3 activity occurs in adult-onset human SLE. However, the amount of Dnase1L3 necessary to prevent lupus onset, if the impact is continuous or requires a threshold, and which phenotypes are most impacted by Dnase1L3 remain unknown. To reduce Dnase1L3 protein levels, we developed a genetic mouse model with reduced Dnase1L3 activity by deleting Dnase1L3 from macrophages (cKO). Serum Dnase1L3 levels were reduced 67%, though Dnase1 activity remained constant. Sera were collected weekly from cKO and littermate controls until 50 weeks of age. Homogeneous and peripheral anti-nuclear antibodies were detected by immunofluorescence, consistent with anti-dsDNA antibodies. Total IgM, total IgG, and anti-dsDNA antibody levels increased in cKO mice with increasing age. In contrast to global Dnase1L3-/- mice, anti-dsDNA antibodies were not elevated until 30 weeks of age. The cKO mice had minimal kidney pathology, except for deposition of immune complexes and C3. Based on these findings, we conclude that an intermediate reduction in serum Dnase1L3 causes mild lupus phenotypes. This suggest that macrophage-derived DnaselL3 is critical to limiting lupus.

immunology↗

bioRxiv2022

Mechanical stimuli activate gene expression via a cell envelope stress sensing pathway

In tissues with mechanical function, the regulation of remodeling and repair processes is often controlled by mechanosensitive mechanisms; damage to the tissue structure is detected by changes in mechanical stress and strain, stimulating matrix synthesis and repair. While this mechanoregulatory feedback process is well recognized in animals and plants, it is not known whether such a process occurs in bacteria. In Vibrio cholerae, antibiotic-induced damage to the load-bearing cell wall promotes increased signaling by the two-component system VxrAB, which stimulates cell wall synthesis. Here we show that changes in mechanical stress and strain within the cell envelope are sufficient to stimulate VxrAB signaling in the absence of antibiotics. We applied mechanical forces to individual bacteria using three distinct loading modalities: extrusion loading within a microfluidic device, compression, and hydrostatic pressure. In all three cases, VxrAB signaling, as indicated by a fluorescent protein reporter, was increased in cells submitted to greater magnitudes of mechanical loading, hence diverse forms of mechanical stimuli activate VxrAB signaling. Mechanosensitivity of VxrAB signaling was lost following removal of the VxrAB stimulating endopeptidase ShyA, suggesting that VxrAB may not be directly sensing mechanical forces, but instead relies on other factors including lytic enzymes in the periplasmic space. Our findings suggest that mechanical signals play an important role in regulating cell wall homeostasis in bacteria. Significance StatementBiological materials with mechanical function (bones, muscle, etc.) are often maintained through mechanosensitive mechanisms, in which damage-induced reductions in stiffness stimulate remodeling and repair processes that restore mechanical function. Here we show that a similar process can occur in bacteria. We find that mechanical stresses in the bacterial cell envelope (the primary load-bearing structure in bacteria) regulate signaling of a two-component system involved in cell wall synthesis. These findings suggest that the mechanical stress state within the cell envelope can contribute to cell wall homeostasis. Furthermore, these findings demonstrate the potential to use mechanical stimuli to regulate gene expression in bacteria.

microbiology↗